Detection of immunoprecipitated systemic lupus erythematosus antigens by immunoblot analysis.
When antigens are isolated from staphylococcal protein A immunoprecipitation pellets for analysis by SDS polyacrylamide gel electrophoresis and immunoblotting, severe background problems, due to the presence of antisera and bacterial proteins, can result. We describe a procedure for the analysis of immunoprecipitated systemic lupus erythematosus antigens (e.g., La, Ro, and Sm) which significantly reduces this background while retaining sensitivity with respect to antigen detection. We have adapted a method previously described (MacSween, J.M. and Eastwood, S.L. Methods Enzymol. 1981; 73:459-471) in which lithium diiodosalicylate is used to separate the immunoprecipitated antigen from a covalent antibody-staphylococcal protein A complex. In addition, a modified series of immunoblot incubations was employed, in which antigenic proteins were identified by incubating blots with the antiserum used for the original immunoprecipitation (e.g., La) followed by protein A-biotin and avidin-alkaline phosphatase. Overall, the procedure is straight-forward and may be applicable to other immunoblot systems.
Rothschild
C B
CB
Wake Forest University Medical Center, Department of Biochemistry, Winston-Salem, North Carolina 27103.
Kilpatrick
B A
BA
D001324:Autoantigens / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D011233:Precipitin Tests; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology; D017411:Ribonucleoproteins, Small Nuclear; D013205:Staphylococcal Protein A; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen
D001324:Autoantigens; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D013205:Staphylococcal Protein A; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human